Cardiac myocyte apoptosis contributes significantly to the development of heart failure. Increased sympathetic nerve activity in the form of norepinephrine is a central feature in patients with heart failure. Norepinephrine, acting via ?-adrenergic receptor (?-AR), induces cardiac myocyte apoptosis. A major goal of my lab is to investigate the molecular signals involved in myocyte apoptosis and myocardial remodeling following ?-AR stimulation and myocardial infarction (MI). The current project is based on our observation that ?-AR stimulation of adult rat ventricular myocytes (ARVMs) increases extracellular levels of ubiquitin (UB; a protein involved in proteasomal degradation of proteins). Using various molecular and biochemical approaches, we provided evidence for a protective role of extracellular UB in ?-AR-stimulated myocyte apoptosis. In human THP1 leukemia cells, CXCR4 is suggested to be a receptor for extracellular UB. Our preliminary data using - i) isolated heart model of global ischemia/reperfusion (I/R; Langendorff model); and ii) in vivo myocardial I/R injury in mice now suggest a cardioprotective role of exogenous UB in myocardial I/R injury. UB treatment decreased infarct size, improved heart function and decreased myocyte apoptosis. In vitro, treatment of ARVMs with UB decreased hypoxia/reoxygenation (H/R)-induced myocyte apoptosis. CXCR4 antagonist, AMD3100, negated the anti- apoptotic effects of extracellular UB. UB activated Akt (anti-apoptotic kinase), while inhibiting H/R-mediated activation of GSK-3? (glycogen synthase kinase-3?; a pro-apoptotic kinase). UB decreased H/R-induced oxidative stress, while maintaining a higher number of ARVMs with polarized mitochondria. UB activated AMPK (a key regulator of cellular energy homeostasis and mitochondrial biogenesis), and increased expression of PGC-1? (a direct regulator of mitochondrial DNA content). UB treatment induced mitochondrial biogenesis, while inhibiting H/R-induced mitochondrial fission. It also inhibited activation of endoplasmic reticulum (ER) stress proteins (PERK and EIF-2?) and expression of Bik (a crucial link between ER stress and mitochondrial fission). These novel observations led us to hypothesize that extracellular UB, most likely acting via CXCR4, plays a protective role in I/R-induced myocyte apoptosis and myocardial remodeling by augmenting mitochondrial biogenesis via the activation of Akt and AMPK, while inhibiting mitochondrial fission via the inactivation of GSK- 3? and ER stress response. Aim 1 will investigate in vivo the role of exogenous UB in I/R-induced cardiac myocyte apoptosis and myocardial remodeling. Aim 2 will identify UB receptor/s in myocytes and heart, and investigate the role of UB receptor/s in the modulation of protective effects of exogenous UB. Aim 3 will test the hypothesis that extracellular UB induces mitochondrial biogenesis via the activation of Akt and AMPK, while inhibiting mitochondrial fission via the inactivation of GSK-3? and inhibition of Bik expression, thereby playing a protective role in H/R-induced myocyte apoptosis. The proposed studies may provide a rationale for the development of cost effective therapeutic use of UB as a treatment for ischemic heart disease.